A small molecule peptide with anti-cancer activity and its application

Abstract

The present invention relates to the field of medicinal chemistry and relates to a small molecule peptide with anti-cancer activity and its application. By analyzing the non-ribosomal peptide synthetase (NRPS) sequence of a Rhodococcus erythropolis strain using bioinformatics software, the possible molecular backbone of the non-ribosomal peptide synthesized by this NRPS was predicted, and a series of small molecule peptides were obtained through chemical synthesis. After activity testing, one of the small molecule peptides was screened out to have a relatively obvious inhibitory effect on the cancer cell line HT29, and it has the value for the development of new anti-cancer drugs.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and relates to a small molecule peptide with anti-cancer activity, as well as its inhibitory effect on the growth of cancer cell line HT29 and its application in the preparation of anti-cancer drugs. Background Art

[0002] Cancer has always been a major health problem faced by mankind, and chemotherapy is an important means of treating cancer. However, most chemotherapy drugs lack specificity, so they will kill normal tissue cells undergoing cell division at the same time, thus often damaging healthy tissues with normal functions. Therefore, discovering new anti-cancer drugs, especially anti-cancer drugs that can specifically target cancer cells, is an important research topic worldwide. Non-ribosomal peptides are a large family of natural products, synthesized by non-ribosomal peptide synthetase (NRPS). Many non-ribosomal peptides have one or more biological activities such as antibacterial activity, siderophore activity, and anti-cancer activity. However, it is quite difficult to isolate and purify the synthetic product from the complex fermentation products of strains; in addition, many gene clusters are nearly silent under most culture conditions, and activating these silent gene clusters requires a large amount of molecular genetics work for different strains.

[0003] With the continuous in-depth study of the structure-function relationship of natural product synthases, bioinformaticians have developed some software (such as antiSMASH, etc.) to predict their synthetic products based on the synthase structure. Researchers combine the prediction results of these software with chemical synthesis methods to develop a new way to discover natural products, namely the synthetic-bioinformatic natural product (syn-BNP) method.

[0004] Based on the whole genome sequencing of a strain of Rhodococcus erythropolis, the present invention uses bioinformatics software to analyze the sequencing results, predicts the possible molecular structure of a non-ribosomal peptide synthesized by NRPS in its genome, and then obtains these small molecule peptides through chemical synthesis. After various biological activity detections, one of the small molecule peptides has an obvious inhibitory effect on the growth of cancer cell line HT29. Summary of the Invention

[0005] To accelerate the discovery of natural products and enrich the natural product molecular library, the present invention uses software to predict the possible molecular structure of a non-ribosomal peptide synthesized by NRPS of Rhodococcus erythropolis, and then obtains these small molecule peptides through chemical synthesis. After detection, one of the small molecule peptides has an inhibitory effect on the growth of cancer cell line HT29.

[0006] A small molecule peptide with anti-cancer activity, chemical formula C31 H 47 N 7 O 6 (Compound 1), with the structural formula as follows:

[0007]

[0008] The small molecule peptide with anti-cancer activity can be applied to the preparation of anti-cancer drugs.

[0009] The small molecule peptide has an inhibitory effect on the growth of cancer cell line HT29.

[0010] Advantages of the present invention:

[0011] In the present invention, a small molecule peptide is obtained by mining a gene cluster of a strain of Rhodococcus. Through anti-cancer experiments on this compound, it is found that it has inhibitory activity against cancer cell line HT29. Therefore, this compound is expected to become a lead compound for anti-cancer drugs and has good development prospects. Description of the Drawings

[0012] Figure 1 is the positive ion mass spectrum of the anti-cancer compound of the present invention

[0013] Figure 2 is the HPLC spectrum of the anti-cancer compound of the present invention Detailed Embodiments

[0014] The following further elaborates the present invention in detail in conjunction with the drawings and specific embodiments.

[0015] The small molecule peptide of the present invention can be directly prepared by the total chemical synthesis of polypeptide technology according to its structural formula. Only one specific synthesis method is provided in this embodiment, and this scheme is only for illustration and not for limitation.

[0016] The preparation process is as follows:

[0017] The small molecule of the present invention is synthesized by solid-phase synthesis technology. First, the N-terminus of the required amino acid is protected with Fmoc and the side chain groups that do not participate in the reaction are protected with acid-sensitive protecting groups. Using the resin as a carrier, the C-terminal amino acid 5 (Trp) is condensed on the resin, and the protecting groups are eluted with an alkaline solution. Then, the above steps are repeated, and amino acid 4 (Leu), amino acid 3 (Val), amino acid 2 (Thr), and amino acid 1 (Orn) are successively condensed on amino acid 5. After the condensation is completed, it is dissociated from the resin and the N-terminal protecting group is removed to obtain the linear polypeptide Orn-Thr-Val-Leu-Trp. After catalyzing its cyclization, the side chain protecting groups are removed to obtain the crude product of the target small molecule polypeptide. The obtained crude polypeptide is purified by HPLC and then lyophilized.

[0018] The finished product was analyzed for purity by HPLC and its molecular weight was confirmed by MS.

[0019] The positive-ion mass spectrum and HPLC chromatogram of the obtained product are shown in Figure 1 , 2 . This small molecule peptide has the effect of inhibiting the proliferation of HT29 cells and is an anticancer compound with application prospects.

[0020] The CCK-8 reagent contains WST-8. In the presence of the electron-coupling reagent 1-Methoxy PMS, this compound can be reduced by dehydrogenases contained in the mitochondria of living cells to generate a highly water-soluble orange-yellow compound - Formazan. The amount of Formazan generated is proportional to the number of living cells. Therefore, for compounds with greater cytotoxicity, the color of the culture medium is lighter. When measuring its light absorption value at a specific wavelength with an enzyme-linked immunosorbent assay (ELISA) reader, the smaller the absorbance value. By calculating the OD value, the survival rate or inhibition rate of the cells can be obtained, thus reflecting the strength of the cytotoxicity of the polypeptide molecule. In the specific examples of the present invention, DMSO was added as a blank control and oxaliplatin was used as a positive control for the anticancer drug, and the IC 50 value of the active compound against human colon cancer cell line HT29 was calculated. Calculated based on the molar concentration in the actual culture medium, the unit is μM.

[0021] The composition of the cell culture medium used in the examples: DMEM culture medium. Before use, 1% penicillin-streptomycin double antibody was added to DMEM, and then 10% ml of fetal bovine serum was added. The cells were cultured at 37 °C and 5% carbon dioxide concentration.

[0022] (I) Structural analysis of the compound

[0023] The small molecule peptide compound 1 of the present invention is a white solid, soluble in dimethyl sulfoxide, methanol, and slightly soluble in water.

[0024] ESI-MS m / e 614.5([M+H] + ).

[0025] (II) Detection of anticancer activity

[0026] 1. Cancer cell line

[0027] Human colon cancer cell line HT29

[0028] 2. Determination of anticancer activity

[0029] The Cell Counting Kit-8 (CCK-8) method was used to detect the anticancer activity of the polypeptide. The specific steps are as follows:

[0030] (1) Preparation of cell suspension: cells grown for 48 h were digested with trypsin and collected. The required concentration was calculated after cell counting. The cells were resuspended and diluted to an appropriate concentration with culture medium for later use.

[0031] (2) Inoculate the prepared cell suspension into a 96-well plate, with 100 μL of cell suspension per well (approximately

[0032] 5×10 4 ), 3 wells of cells in each sample were repeated experiments.

[0033] (3) After inoculation, the cells were cultured in a 37°C incubator for 24 hours.

[0034] (4) Add 10 μL of peptide solution of different concentrations or DMSO or oxaliplatin for control to each well.

[0035] (5) After adding the drug, the cells were placed in an incubator at 37°C for 24 hours.

[0036] (6) Add 10 μL of CCK-8 solution to each well. When adding, the tip of the pipette should be slightly immersed in the culture medium.

[0037] After adding the reagents, tap the culture plate gently to help mix. (Or prepare 10% CCK-8 solution for replacement)

[0038] (7) Continue to culture in the incubator at 37°C for 4 hours.

[0039] (8) The absorbance was measured in an ELISA instrument using a dual wavelength, with the detection wavelength being 450 nm and the reference wavelength being 600 nm.

[0040] (9) Calculate cell survival rate and inhibition rate based on absorbance.

[0041] Cell survival rate = [(As-Ab) / (Ac-Ab)] x 100%

[0042] Inhibition rate = [(Ac-As) / (Ac-Ab)] x 100%

[0043] As: absorbance of the experimental well

[0044] Ac: absorbance of control well

[0045] Ab: absorbance of blank well

[0046] After obtaining the cell survival rate after adding small molecules of different concentrations, the cell survival rate curve corresponding to the final concentration of small molecules in the culture medium was calculated. Finally, the small molecule concentration when the cell survival rate was 50% was calculated as IC 50

[0047] Table 1. Inhibitory Activity of Compounds against HT29 Cancer Cell Line

[0048] Compound Compound 1 Oxaliplatin <![CDATA[IC 50 (μM)]]> 16.75 1.62

[0049] Experimental Conclusion: The antibacterial compound of the present invention has obvious inhibitory activity against human colon cancer cell line HT29. The control group, oxaliplatin, is a mature commercial anti-cancer drug. The IC50 value of the small molecule peptide of the present invention is 10 times that of this product without further optimization; although there are some differences in activity, it still has great development potential. Moreover, compared with traditional anti-cancer drugs, the synthesis of polypeptides is simpler and the cost is lower. Therefore, the small molecule polypeptide of the present invention can be used as a lead compound for anti-cancer drugs and play a guiding role in the development of subsequent anti-cancer drugs.

[0050] The content of the present invention is not limited to what is listed in the specific embodiments. Any equivalent transformation of the technical solution of the present invention adopted by those of ordinary skill in the art by reading the specification of the present invention is covered by the claims of the present invention.

Claims

1. A small molecule peptide with anti-cancer activity, Characterized in that, Its chemical formula is C 31 H 47 N 7 O 6 , and its structural formula is as follows: 。 2. Use of the small molecule peptide according to claim 1 in the preparation of an anti-colorectal cancer drug.

3. An anti-cancer drug, Characterized in that, It contains the small molecule peptide according to claim 1.

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